Due to poor outcomes of patients with invasive fungal infections and expensive health care needs of these patients, invasive fungal infections carry exceedingly high human and financial costs. The cost of invasive fungal infections to the healthcare system is conservatively estimated to be $2.6 billion annually in the US alone. Current antifungal agents can have limited clinical efficacy, are occasionally toxic, and are increasingly ineffective due to emerging resistance. Despite this need, no completely novel class of antifungals has been developed in over 10 years. This project seeks to address the need for new therapies by targeting a signal transduction network required for invasive fungi to survive in humans. Calcineurin is a highly conserved protein phosphatase that is important in mediating cell stress responses and necessary for invasive fungal disease. Currently available calcineurin inhibitors cyclosporine A (CsA) and tacrolimus (FK506) are active in vitro against the major invasive fungal pathogens, but they are also immunosuppressive in the host, limiting therapeutic effectiveness. The objective of this Phase I SBIR project is to identify non-immunosuppressive CsA analogs with antifungal activity against clinically relevant fungal species, either active alone or in combination with existing antifungal drugs. The project is enabled by one of the largest known collections of CsA analogs bearing modifications at multiple sites that introduce both chemical and biological diversity. Using that collection, preliminary studies have already defined CsA analogs that have substantially lower or no immunosuppressive action, yet retain effective antifungal activity in vitro against the two most common pathogenic fungi, Candida albicans and Aspergillus fumigatus. This proposal will identify additional CsA analogs with low immunosuppression and high antifungal potency against both species, including clinically-relevant drug resistant strains. Active analogs will be tested against difficult to treat species, such as C. krusei, C. glabrata, and A. terreus, to bette define the spectrum of antifungal activity. Active compounds will also be evaluated for in vitro cytotoxicity and metabolic stability, to identify up to 10 candidates suitable for future testing i animal efficacy models at the outset of Phase II. Additional Phase II studies will include testing against a wider spectrum of clinical isolates, evaluation against biofilms, and chemical optimization for selection of preclinical lead candidate(s) for ultimate clinical evaluation.